Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH) consisting of three domain, CH1, CH2 and CH3). CH1 and CH2 of the heavy chain are separated from each other by the so-called hinge region. The hinge region normally comprises one or more cysteine residues, which may form disulphide bridges with the cysteine residues of the hinge region of the other heavy chain in the antibody molecule.
Recently, antibodies have become a major focus area for therapeutic applications, and many antibody drug products have been approved or are in the process of being approved for use as therapeutic drugs. The desired characteristics of therapeutic antibodies may vary according to the specific condition which is to be treated. For some indications, only antigen binding is required, for instance where the therapeutic effect of the antibody is to block interaction between the antigen and one or more specific molecules otherwise capable of binding to the antigen. For such indications, the use of Fab fragments, the only function of which is to bind antigen, may be preferred. For other indications, further effects may also be required, such as for instance the ability to induce complement activation and/or the ability to for instance bind Fc receptors, protect from catabolism, recruit immune cells, etc. For such use, other parts of the antibody molecule, such as the Fc region, may be required. Some full-length antibodies may exhibit agonistic effects (which may be considered to be undesirable) upon binding to the target antigen, even though the antibody works as an antagonist when used as a Fab fragment. In some instances, this effect may be attributed to “cross-linking” of the bivalent antibodies, which in turn promotes target dimerization, which may lead to activation, especially when the target is a receptor. In the case of soluble antigens, dimerization may form undesirable immune complexes.
For some indications, monovalent antibodies may thus be preferable. The presently available Fab fragments show inferior pharmacokinetics due to their small size resulting to filtration in the kidneys as well as their inability to interact with the Brambell receptor FcRn (Junghans R P et al., Proc Natl Acad Sci USA 93(11), 5512-6 (1996)), therefore being unstable in vivo and having very rapid clearance after administration.
There is thus a need for stable monovalent antibodies which can be used as therapeutics.
Dimeric, monovalent antibodies (Fab/c), wherein the Fc region comprises two Fc polypeptides, have been described (WO200563816 to Genentech and Parham P, J. Immunol. 131(6), 2895-902 (1983).
Ig half-molecules, which have a dimeric configuration consisting of only one light chain and only one heavy chain, have been described as the result of rare deletions in human and murine plasmacytomas. Studies on the biochemical nature of these half-molecules showed that they consist of IgG1 molecules in which the heavy chain CH1, hinge and CH2 regions appeared normal, whereas deletions were found in the CH3 region. The mutations appeared to be located in CH3 and the hinge peptide appeared normal (Hobbs, J R et al., Clin Exp Immunol 5, 199 (1969); Hobbs, J R, Br Med J 2, 67 (1971); Spiegelberg, H L et al., Blood 45, 305 (1975); Spiegelberg, H L et al., Biochemistry 14, 2157 (1975); Seligmann M E et al., Ann Immunol (Paris) 129C, 855-870 (1978); Gallango, M L et al., Blut 48, 91 (1983)). It was also showed that this human IgG1 half-molecule is rapidly catabolized (half-life in man was 4.3 days) and, in monomeric form, is unable to bind C1q or Fc receptors on human lymphocytes, monocytes or neutrophils (Spiegelberg, H L. J Clin Invest 56, 588 (1975)).
Murine IgA half-molecules which were generated by somatic mutation have also been described (Mushinski, J F, J Immunol 106, 41 (1971); Mushinski, J F et al., J Immunol 117, 1668 (1976); Potter, M et al., J Mol Biol 93, 537 (1964); Robinson, E A et al., J Biol Chem 249, 6605 (1974); Zack, D J et al., J Exp Med 154, 1554 (1981)). These molecules were shown to all contain deletions of the CH3 domain or mutations at the CH2—CH3 boundary.
WO2007059782 (Genmab) describes human monovalent antibodies comprising a light chain and a heavy chain, wherein
a) said light chain comprises the amino acid sequence of the variable (VL) region of a selected antigen specific antibody and the amino acid sequence of the constant (CL) region of an Ig, and wherein, in case of an IgG1 subtype, the amino sequence of the constant (CL) region has been modified so that it does not contain any amino acids capable of participating in the formation of disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the constant (CL) region of the Ig, in the presence of polyclonal human IgG or when administered to an animal or human being, and
b) said heavy chain comprises the amino acid sequence of the variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the constant (CH) region of human Ig, wherein the amino acid sequence of the constant (CH) region has been modified so that the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not contain any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the constant (CN) region of the human Ig, in the presence of polyclonal human IgG or when administered to an animal or human being.
As shown in WO2007059782, these monovalent antibodies have a more favorable in vivo half-life than Fab fragments. WO2008145140 describes variants of these monovalent antibodies wherein intermolecular CH3-CH3 interactions are destabilized. The present application describes alternative and improved variants of the monovalent antibodies disclosed in WO2007059782 and WO2008145140. These variants remain monovalent even under conditions that favor intermolecular CH3-CH3 interactions.
Human IgG4 molecules exist in various molecular forms which differ by the absence or presence of inter-heavy chain disulphide bonds located in the hinge region. Thus IgG4 molecules exist in which two, one or no inter-heavy chain disulphide bonds have been formed (Schuurman, J. et al., Mol Immunol 38, 1 (2001)). Under physiological conditions, these molecular forms of IgG4 may be in equilibrium with each other. Human IgG4s exist as tetramers in solution consisting of two Ig heavy and two light chains, as common for immunoglobulin G molecules, irrespective of the absence or presence of these interchain disulphide bonds (Schuurman 2001 supra; Gregory, L. et al. Mol Immunol 24, 821 (1987)). Only upon denaturation under non-reducing conditions, the two non-covalently associated half-molecules dissociate as demonstrated by size-determination analysis such as SDS-PAGE (Schuurman, J. et al. Mol Immunol 38, 1 (2001); Deng, L. et al. Biotechnol Appl Biochem 40, 261 (2004)). It has been shown that mutation of the residues of the hinge region which are involved in inter-chain disulphide bond formation or deletion of the hinge region lead to creation of a homogeneous pool of IgG4 molecules in solution, which pool consists of tetrameric molecules consisting of two light chains and two heavy chains (Schuurman, J. et al. Mol Immunol 38, 1 (2001); Horgan, C. et al. J Immunol 150, 5400 (1993)). The IgG4 hinge-deleted and mutated antibodies also demonstrated an improved capability of antigen crosslinking when compared to native IgG4 molecules (Horgan, C. (1993) supra).
It has been shown that administration of two recombinant monoclonal IgG4 antibodies having different antigen-binding specificities to a mouse leads to in vivo formation of bispecific antibodies. The phenomenon can be reproduced in vitro by incubating IgG4 antibodies with cells or under reducing conditions. It has been shown that IgG4 antibodies having different antigen-binding specificities engage in Fab arm exchange which is stochastic and in which all IgG4 molecules seem to participate. Thus, IgG4 antibodies form bispecific antibodies without concomitant formation of aggregates.
IgG4 antibodies therefore have unusual properties which are undesirable in vivo: IgG4 antibodies are unstable, dynamic, molecules which engage in Fab arm exchange. An administered therapeutic IgG4 antibody may exchange with endogenous IgG4 antibodies with undesired specificities. The random nature of this process introduces unpredictability which is highly undesirable for human immunotherapy.
In one aspect, the present invention relates to stabilized forms of IgG4 antibodies that have a reduced ability to undergo Fab-arm exchange. Stabilized forms of IgG4 have previously been described in WO2008145142 (Genmab). It has now surprisingly been found that specific alternative substitutions in human IgG4 can prevent Fab arm exchange, and thus stabilize IgG4.
In summary, the present invention relates to positions in the constant region of antibodies, in particular the CH3 region of IgG4, which affect the strength of CH3-CH3 interactions. Mutations that either stabilize or destabilize this interaction are disclosed herein.
When introduced in the monovalent antibody context described in WO2007059782, the destabilizing mutations contribute to keeping the antibodies monovalent even under conditions that favor intermolecular CH3-CH3 interactions. When introduced in the IgG4 context, the stabilizing mutations contribute to preventing undesired Fab arm exchange.